Annex 1. Project Report to DEFRA.

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DEFRA commission PS2108

Towards cost effective registration of natural semiochemicals for crop protection in the UK

Report for distribution and discussion with PSD

Authors: Rothamsted Research, and JSC International Ltd.

Date: 10th August 2005

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Contents

Contents ...................................................................................................................................... 2 1. Introduction ......................................................................................................................... 3 2. Literature searching ............................................................................................................ 6

2.1 Chemical Search Terms:.................................................................................................62.2 Additional Search Terms:...............................................................................................62.3 Databases searched.........................................................................................................72.4 Private databases and begging for data...........................................................................92.5 Data storage:.................................................................................................................102.6 Obtaining reprints.........................................................................................................112.7 Translation....................................................................................................................11

3. Data collation .................................................................................................................... 11 3.1 Abstracting data into Tier II documents.......................................................................113.2 Specific Points on Completing Dossier Summaries.....................................................12Section 1 Identity................................................................................................................12Section 1 Physical and chemical properties........................................................................13Section 2 Analytical methods.............................................................................................15Section 3 Toxicological and metabolism studies................................................................15Section 4 Residues..............................................................................................................19Section 5 Fate and behaviour in the environment...............................................................19Section 6 Ecotoxicological studies.....................................................................................20Classification and labelling...................................................................................................24

4. Assessment of costs required to complete the packages to a standard suitable for regulatory purposes ................................................................................................................... 24 Appendix 1 - Use scenarios used as the basis of the data gap analysis .................................... 27 Appendix 2 Summary of costs outstanding to complete the packages for regulatory submission ................................................................................................................................. 31

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1. Introduction

Naturally occurring semiochemicals, that control the behaviour or development of pests and their hosts, can be extracted from cultivated non-food plants and deployed for the protection of larger areas of crop plants. However, naturally derived semiochemicals, that are applied as “plant protection products”, require registration under EC Directive 91/414/EEC.

In the United Kingdom, there is considerable demand for use of alternatives in crop protection to conventional synthetic broad-spectrum pesticides which have a predominantly toxic mode of action on the target pest. Since semiochemicals act as signals regulating behaviour or development of the pest (or crop), rather than by toxic or other direct physiological effects, effective pest control methods based on their use may be more acceptable not only over synthetic conventional pesticides but also over botanically-derived toxicants. Although it is also ‘conventional wisdom’ that many do not have very good or predictable efficacy; semiochemicals can be valuable components of integrated pest management programmes particularly because their risk profiles can be favourable in comparison with conventionally available synthetic chemicals (e.g. due to their specific mode of action combined with low effective use rates), and they may also be acceptable in organic production systems.

However, the specific profile of the candidate semiochemicals which are proposed for registration and subsequent commercialisation must be demonstrated objectively in the formal assessment of potential hazards and risks made by the Pesticide Safety Directorate (PSD) based on information contained in a registration dossier. The developing industry has indicated that data requirements, their presentation and assessment procedures are based too heavily on procedures for conventional pesticides and thus appear a potential barrier to commercialisation, and hence the availability, of more desirable control methods. The publication of information arising from pilot registration exercises would assist this emerging industry with development of appropriate registration packages and further exploitation of semiochemicals. This would in turn contribute to addressing the demand for pest control systems which exploit natural processes e.g. biorational alternatives which may be preferable to some conventional methods and currently available GM technologies.

The main objective of this research project was be to take information on three specific examples of semiochemicals and assess the availability of data and produce packages suitable for registration, presenting this information in the dossier format required under 91/414/EEC which is acceptable to regulatory authorities. The main activities involved providing data for long term previous and current human contact with the three natural semiochemicals, together with toxicological data as available for each, or for closely related natural products, and any appropriate synthetic analogues. Any further data required will be provided by ongoing research in other projects, with this study concentrating on the compilation of existing data, and those from other sources, and the computation of or extrapolation from these data for the purposes of the registration packages.

Europe has adopted the Organisation for Economic Co-operation and Development (OECD) harmonised approach where much of the core chemical data set can be reduced for semiochemicals1 that affect the behaviour of arthropods. Further reductions in data

1 Defined by OECD as: “Semiochemicals (SC) are chemicals emitted by plants, animals, and other organisms - and synthetic analogues of such substances - that evoke a behavioural or physiological response in individuals of the same or other species. They include pheromones and allelochemicals. This report pertains only to SCs that affect the behaviour of arthropods.”

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requirements are possible for Straight-Chained Lepidopteran Pheromones (SCLPs) which are pheromones with a well-defined unbranched aliphatic structure, characteristic of the Lepidoptera. Thus it was considered of value to consider the OECD semiochemicals guidance document2 while compiling the draft dossier documents in this project. The guidance was useful in some cases but of limited value (and occasionally misleading) at times for these non-SCLP substances.

OECD have compared the inherent properties of semiochemicals with properties of conventional chemicals. It is considered that arthropod semiochemicals products pose lower potential risk to human health and the environment than conventional pesticides for a number of reasons. All these factors minimise the risk of adverse effects from the use of semiochemicals:

They act by modifying behaviour of the pest species rather than killing it They are more target specific than conventional insecticides They are generally effective at very low rates so can be used at concentrations close to

those in nature They are generally volatile and usually dissipate rapidly in the environment thus

minimising residues in crops and exposure of non-target organisms End use products may be used in dispensers that result in little direct exposure to humans

and non-target organisms Furthermore, SCLPs are considered to be of low toxicity to mammals.

Three natural semiochemicals were chosen and a probative registration package assembled for each, to devise a general framework. These were:

1. crops. Hop β-acids. This material is extracted from brewing hops and acts as an effective antifeedant (a semiochemical interfering with feeding behaviour) against, for example, the two-spotted spider mite, Tetranychus urticae, a pest of high value horticultural

2. Methyl salicylate. This is employed as the major component of a fully definable essential oil (oil of wintergreen) and acts as an indicator of plant stress, and therefore non-host status, thereby reducing aphid colonisation of, for example, cereal crops.

3. cis-Jasmone. This is obtained as a major component of various essential oils and acts as a plant signal, or plant activator, that usefully “switches on” plant defence and reduces thereby, for example, aphid numbers on cereal crops.

The milestones for the project were:

1. Search available literature and databases for all available data for the three semiochemicals. (end of month 6)

2. Collate data under EC directive 91/414 format, conduct a data gap analysis

and define areas where further research is required. (end of month 8)

2 OECD Environment, Health and Safety Publications. Series on Pesticides No. 12. Guidance for Registration Requirements for Pheromones and Other Semiochemicals Used for Arthropod Pest Control

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3. Construct and maintain a web site, which will incorporate the data relevant to each registration package as it is assembled. (end of month 7)

4. Assemble example registration packages for the three semiochemicals and

submit to PSD for scrutiny. end of month 12)

The project commenced on 01/01/2004.

There were two objectives in the project; to develop registration dossiers for three semiochemicals and also to comment on the challenges encountered in attempting to do this using data from the public domain. The documents produced which are summarised in the table below are not forming a submission to PSD at the current time, but they will be used as the basis of a future regulatory submission.

The following table summaries the outputs according to each milestone.

Milestone Who Outputs1. Search available literature and

databases for all available data for the three semiochemicals

RR / JSC Microsoft Access database format summarising all relevant papers found.Comments on the procedure used are presented in this report.Reports found were collated as below.

2. Collate data under EC directive 91/414 format…

RR / JSC Document K-II Sections 1-6 Reference lists Sections 1-6

…conduct a data gap analysis… JSC Findings presented in this report and JSC overview comment sheets associated with each M-II dossier document.

…and define areas where further research is required

JSC Findings presented in this report with comments on costings to complete the dossiers to submission standard.

3. Construct and maintain a web site, which will incorporate the data relevant to each registration package as it is assembled

RR A draft format of a web site has been produced and a final version will need to be agreed by RR, JSC and PSD

4. Assemble example registration packages for the three semiochemicals and submit to PSD for scrutiny

RR / JSC Document K-II Sections 1-6 Reference lists Sections 1-6

RR / JSC Document D-1 GAP formsRR / JSC Draft Document M-II Sections 1-6, plus:JSC -- Additional input to complete the methyl

salicylate M-II, Section 3 (Toxicology section)

RR: Rothamsted ResearchJSC: JSC international Ltd.

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The documents generated and listed above will be of value alongside this report as worked examples to form the basis of discussions with PSD, and complete the project. We hope that the experiences gained compiling these three examples will allow productive discussion on potential ways forward for the regulatory process for semiochemicals and plant extracts.

2. Literature searching

As wide a selection of databases possible, both free and by subscription were searched. Private databases such as the Research Institute for Fragrance Materials (RIFM) and the Flavor and Extract Manufactures Association (FEMA) database were a different matter (see below). All databases were searched using a range of keywords, however, with cis-jasmone and hop -acids the references were usually few, so that the databases were searched using chemical terms only and relevant hits found by manual searching the output. Search terms were adjusted to suit the requirements or content of the database.

2.1 Chemical Search Terms:

Semiochemical Search Terms

Methyl salicylate: methyl salicylate; methyl AND salicylate (Boolean); methyl (1W) salicylate (word proximity); salicylate; salicylic; wintergreen; 119-36-8 (CAS number).

cis-Jasmone: cis-jasmone; (Z)-jasmone; Z-jasmone; jasmone; jasmon*; cis AND jasmone (Boolean); 488-10-8 (principal CAS number).

Hop -acids: hop beta-acids; (hops OR hop) AND beta AND acids (Boolean); hops; lupulone; 468-28-0 (CAS number); lupulon; lupulone OR lupulon (Boolean); colupulone; adlupulone; prelupulone; postlupulone.

2.2 Additional Search Terms:

The majority of the following additional search terms were suggested by JSC International. These were abbreviated using wild cards (* or ?) as shown where the database supported it. Where it did not, the terms were searched individually, e.g. toxicity or toxicology. Toxicology/human exposure, safetytoxic*; human; medical; poison*; rat; LD50; repro*; diet*; expos*; operator?; worker?; farm*; material safety data sheet; msds.

Environmental exposure/breakdownenviron*; groundwater; water; ecotox*; bird; worm; earthworm; daphnia; bird; plant; metab*; decomp*; degrad*.

Physico-chemical propertiesphotolysis; hydrolysis; melting; UV; NMR; solubil*; mass AND spectr* (Boolean); vapo?r; logKow; flamm*.

2.3 Databases searched

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Databases were searched in the following order:

1. Google http://www.google.com/. Google uses non-Boolean searches (assumes AND) but the advanced option can be used to format searches more precisely. I would always recommend searching Google before going on to more specialised databases. It is a remarkably powerful search engine that is capable of finding one obscure search term on page 49 of a 100 page pdf document! Several very useful secondary sources, with references were originally located using Google, for example:

Methyl salicylate: California Environmental Protection Agency (EPA) Department of Pesticide Regulation, Medical Toxicology Branch Methyl Salicylate: Data Gap Status.

Methyl salicylate: National Institute for Occupational Safety and Health: Registry of Toxic Effects of Chemical Substances. Salicylic acid, methyl ester.

National Institute for Occupational Safety and Health: Registry of Toxic Effects of Chemical Substances. 2 - Cyclopenten - 1 - one, 3 - methyl - 2 - (2 - pentenyl) - , (Z) –(cis-jasmone)RTECS #: GY7301000

National Institute for Occupational Safety and Health: Registry of Toxic Effects of Chemical Substances. Salicylic acid, methyl ester.

UIC/NIH Center for Botanical Dietary Supplement in Women's Health. (Contains chemical structures and physical properties of hop alpha-acids, hop beta-acids and iso-alpha acids.).

Safety assessment of salicylic acid, butyloctyl salicylate, calcium salicylate,C12-15 alkyl salicylate, capryloyl salicylic acid, hexyldodecyl salicylate, isocetyl salicylate, isodecyl salicylate, magnesium salicylate,MEA-salicylate, ethylhexyl salicylate, poassium salicylate, methyl salicylate, myristyl salicylate, sodium salicylate, TEA-salicylate, and tridecyl salicylate.

Committee for veterinary medical products: Salicylic acid, sodium salicylate, aluminium salicylate, basic and methyl salicylate: Summary report.

2. Scirus http://www.scirus.com/. Scirus is the best non-subscription web-based search engine for scientific papers. It also searches entries in BioMed Net, Medline, PubMed and Science Direct and so saves searching those separately. It searches 167 million web pages. You can specify whether to search web sources or journal sources (or both). It uses Boolean input and has more options in advanced search. Earliest papers date from the 1950s but coverage is thin for early 20th century information. Output is ranked by relevance. One needs to register with Science Direct and BioMed Net to access full text articles (in some cases) although the summary is always accessible.

3. Web of Knowledge (WOK) (formerly Web of Science) http://wok.mimas.ac.uk/. Subscription database open to universities, colleges and institutes only and accessed via Athens account. It originally only had references to articles from 1981 to date but now purports to include papers back to 1945, but coverage of early years is patchy. WOK uses Boolean searching in separate database fields. Earlier papers often don’t have an abstract. It can do useful things like citation searching.

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4. Beilstein http://crossfire@mimas.as.uk. is primarily a very powerful chemical database but is now expanded to include pharmacological data, although their inclusion seems patchy. Data input is by name, name fragment, chemical structure or chemical structure fragment. Beilstein has useful points like “LitLink” in which the web link to the reference is made directly. This was very useful in finding physical properties. Contains early references (19th century), as it is derived from the original Beilstein Handbook. Subscription only and accessed via Athens account. There were many more hits for the pharmacological data for methyl salicylate when I searched the database in June than in January, suggesting that more data are being entered retrospectively. There were little data apart from basic chemical information for cis-jasmone and lupulone and its analogues, although I had found them by using other databases.

5. Edina Biosis http://biosis.edina.ac.uk/. Biological abstracts. Boolean searching in database fields. Useful for purely biological and toxicological searching. Accessed via Athens account.

6. CAB Abstracts http://gateway.ut.ovid.com/. This database covers international journals in agriculture, forestry, and allied disciplines in the life sciences and from 1973 to current. Accessed via Athens account. Boolean searching in fields. Very few useful hits were found.

7. Toxnet. http://toxnet.nlm.nih.gov/. This site searches ten toxicological databases, including Toxline. Boolean searching in an index field. Rothamsted subscribes to this database.

8. PAN Pesticide database – chemicals. http://pesticideinfo.org/. This covers Identification, toxicity, use, water pollution potential, ecological toxicity and regulatory information on pesticides and their active ingredients. Searching is by chemical name, common name, name-fragment or CAS number. There are useful links to regulatory web sites. “Oil of Wintergreen” was located on this database, however, no new data were located that hadn’t already been found. Interestingly, octyl salicylate was also listed.

9. Dialog Web. http://www.dialogweb.com This searches over 600 databases. Rothamsted subscribed for me for three months. The following were searched:

34 and 434 (Sci search), 94 (Japanese science and technology (JICST)), 245, waternet, (CAS), 306 (pesticide fact file), 235 (agroprojects), 10 (Agricola), 156 (Toxfile), 70 (SEDBASE), 161 (occupational safety and health), 229 (drug information full text), 172 (Embase alert), 307 (dictionary of substances and their effects) 317 (chemical safety news base), 331 (material safety data sheets), 337 Registry of toxic effects of chemical substances ((RTECS)), 337 (Chemtox).

Dialog Web was generally the least productive, as surprisingly almost no new relevant information was found that had not already been previously located.

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The European Chemical Substances Information System was checked for the three compounds3 and an IUCLID Dataset was found for methyl salicylate (CAS 119-36-8), thus indicating that in parallel, notifiers are defending the substance in the EU for a range of uses.

The only other database that could have been searched but was not was STN express (CAS online) as it was considered that its coverage had already been adequately searched.

2.4 Private databases and begging for data

The most obvious and relevant database is the RIFM and FEMA database. Response to letters asking for access elicited the following (typical) reply:

“Philip,In response to your e-mail requesting data for methyl salicylate and cis-jasmone, we do not distribute our company data (ex. Moreno and Epstein reports) unless requested by a governmental organization or by a RIFM member. You are able to purchase electronic copies of our monographs for those materials if you are interested, though please note they have not been updated since their respective publication dates.If you have access to a library, the monographs for those materials were published in special issues of Food and Chemical Toxicology in 1979, v17, p845, Special Issue V and 1978, v16, p821, Special Issue IV.

Sincerely,Christen Sachse-VasquezTechnical ManagerResearch Institute for Fragrance Materials, Inc.50 Tice BoulevardWoodcliff Lake, NJ 07677Phone: 201-689-8089 ext. 107FAX: 201-689-8090csachse-vasquez@rifm.org”

Unfortunately, as I already had the special issues of Food Chem. Toxicol. that were mentioned and I knew they only contained bottom line summary data.

Contacting the United States Food and Drug Administration (US FDA) directly as below on a different topic elicited no response at all.

“Dear Sir,

I am currently involved in an exercise to determine what information is available to register a number of semiochemicals as crop protection agents within the European Union. This includes assembling available data on the toxicology of these substances. Two of the compounds in which we are interested are methyl salicylate and cis-jasmone. Two reports that I am particularly trying to find are:

3 The European Chemical Substances Information System (ESIS) is found on the European Chemicals Bureau website http://ecb.jrc.it/

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LaWall and Harrison Research Labs. (1964) Study on of methyl salicylate on rats (L&H58188)

Abbot and Harrison (nd) Studies on osseous changes in the rat and mouse and liver and kidney effects in the dog.

I understand that both reports were submitted to the FDA in 1998 in response to a Freedom of Information (FOI) request.

I would be grateful if you could let me know if these reports are available for purchase and if you have any other toxicology reports on methyl salicylate or cis-jasmone that we can buy.”

[sent 27/05/04] I eventually contacted Dr Steve Meakins (Steve.Meakins@questintl.com) at Quest International, Ashford, Kent and asked if he could search the RIFM-FEMA database. This he did and faxed me the information. There were seven relevant and new reports, but unfortunately they only had summary data. I was unable to obtain the non-public domain reports and had to resort to including the summary data in the dossiers.

2.5 Data storage:

All references were held in a flat-format MS-Access database containing the following fields:

Field Data Type Notes

Ref. No. numeric A unique reference number, auto-entered.Annex no. text The annex number used in the tier II dossiers.Semiochem. text A choice field with “cis-jasmone”, hops beta acids” and

“methyl salicylate”.Title textAuthors textJournal textYear numericVolume numericPart textPages textKey words textWeb site textDate date The date when the web site was last updated, if known.Abstract memo Either, the abstract of the paper was copied and pasted

or scanned in or, if important, a new summary was written.

It was found convenient to export fields from selected records into Excel and then into a MS-Word table to obtain the correct format prior to incorporation in to the tier II reference lists. All interesting references were incorporated into the database, however, of the 202 (cis-jasmone 19, hop beta-acids 20 and methyl salicylate 163) papers, reports and web sites, only 80 (cis-jasmone 10, hops beta acids 9 and methyl salicylate 61) were used in the dossiers. The majority of those not incorporated were articles used as secondary sources (references) to locate primary information. Obviously, a further source of information was the reference lists

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of the documents. Irritatingly, some reports produced by government agencies (e.g. the EPA) were found to contain only summary information and no references or indication of where the information was obtained.

2.6 Obtaining reprints

All papers and documents not in the Rothamsted library or in journals to which Rothamsted does not have on-line access were obtained through inter-library loan via the library staff. Our library personnel were extremely helpful and obtained several reprints and reports from some very obscure journals. It is difficult to see how this exercise could be performed without dedicated high-class library services.

2.7 Translation

A number of the published papers obtained are not written in English. Based on the current requirements of PSD, it is understood that these would need to be translated into English, which is identified as an additional cost.

3. Data collation

3.1 Abstracting data into Tier II documents

The author preparing the dossier sections is an experienced pesticide scientist with experience in regulatory submissions which would be comparable to a typical biocontrol applicant. This author conducted the literature searching and summarised the studies found in Tier 2/M-II document style for the purposes of producing a dossier for each compound. This approach best allowed the clear elucidation of the challenges encountered in preparing submissions on semiochemical products from the literature.

The primary problem encountered in summarising papers for the Tier II dossier documents (M-II, Sections 1-6) was a difficulty in understanding the degree of information in the summary that was needed. The examples in the initial JSC guidance document are GLP-format company reports and quite different from the open literature papers that we had to deal with. Quite often, for example in the papers describing the field efficacy of these semiochemicals, the results data were presented in graphical format, rather than hard tabular data with statistics. The former are fine when presenting the overall trends of the results to the journal reader but not much use when trying to calculate the percentage control produced by a specific treatment. All of the data were non-GLP, with the exception of two US papers on the reproductive toxicity of methyl salicylate. Consequently, the standard of data varied considerably. In some cases there was no experimental methodology at all, just the bland statement that the oral LD50 on rats was a certain value. In general, what information was not mentioned in the document, e.g. strain, sex, weight, but is required in a study of this type was specifically stated in the dossier summary to be unreported. This was included in response to the feed-back from JSC on the original draft dossier for methyl salicylate. Most studies were non-standard and the rat feeding studies for methyl salicylate located were over non-standard periods of time. It was often necessary to include bottom line data with no experimental information at all, as frequently that was all the information available.

There was much more information on methyl salicylate, as a consequence of its being used as a flavouring material and pharmaceutical, than the other two materials. The fact that the

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material has been used in the home for many years and the occurrence of many fatal and non-fatal poisoning incidents has catalysed the undertaking of toxicological studies. However, the fact that a material has been used in perfumery (cis-jasmone) or imbibed in beer (hop -acids) for many years without any reported hazards has not necessarily been correlated with the expected level of published safety data, although both materials have been granted GRAS (Generally Recognised As Safe) status by the US-EPA. It is clear that regulatory authorities are now aware of the dearth of toxicological information extant on some older substances (e.g. pharmaceuticals) and there are moves afoot to correct this, for example see: “The flavor and fragrance high production consortia: the aromatic consortium: test plan for benzyl derivatives”. This is essentially a data gap analysis on the toxicological information for methyl salicylate. The EPA has also published a data gap analysis on the toxicological information on methyl salicylate (California EPA Department of Pesticide Regulation, Medical Toxicology Branch Methyl Salicylate: Data Gap Status. and NIOSH a toxicological study for both methyl salicylate and cis-jasmone (National Institute for Occupational Safety and Health: Registry of Toxic Effects of Chemical Substances. Salicylic acid, methyl ester and National Institute for Occupational Safety and Health: Registry of Toxic Effects of Chemical Substances. 2 - Cyclopenten - 1 - one, 3 - methyl - 2 - (2 - pentenyl) - , (Z) -).

3.2 Specific Points on Completing Dossier Summaries

Since this project is considering actives that are in the pre-commercial phase; formulations of the active substances are under refinement. As such, the evaluation was based around core Annex II data i.e. that relating to the active substance. The requirements for Annex III data on the formulated products along with the technical specification of the technical material as manufactured will be further clarified when the commercial phase of development occurs.

For the semiochemicals studied (methyl salicylate, cis-jasmone and hop -acids), it is possible to use a regulatory approach that is slightly different from that which would be used for conventional chemical pesticides. For conventional chemicals data are generated on the chemical itself followed by data on the formulation and these are then used, with exposure information, to assess the risks posed by the use of the plant protection product. By changing this emphasis slightly and focusing on the exposure profile of these semiochemicals from the start it is possible to generate a dossier that maximises the use of a limited data package. This approach could be considered acceptable because of the specificity, source, origin and occurrence of the chemicals under consideration.

In view of the potential for using such an approach which focuses on the exposure profile early in regulatory development, a significant overlap has become apparent between the Annex II (active substance) and Annex III (formulation) sections of the dossier. Final commercial formulation details are not available for all the actives under consideration in this project, but it was been possible, and was considered necessary to focus on representative use scenarios which are presented in Good Agricultural Practice (‘GAP’) tables (Appendix 1).

The following sections discuss the issues noted when compiling each dossier section from published literature.

Section 1 Identity

There was no problem completing the basic data, although hop -acids, being a mixture of closely related analogues was most effectively compiled as a table. Except for hop -acids,

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which is produced by Botanix, there is no information on manufacturing or analysis for the other two materials as they are commodity chemicals. Consequently, there was little or no information available on the proposed technical specification of the technical material intended for use in formulated products or whether any toxicologically relevant impurities had been identified. Ultimately, this section will need to address the profile of the commercial materials and those materials used to generate data within the published reports. This is critical to allow and support the use of the wide range of publicly available information found in this exercise to support a registration.

Section 1 Physical and chemical properties

More physical-chemical properties were found for methyl salicylate than for cis-jasmone and hop -acids.

Most of the data included in this section of the draft dossiers had been found using Beilstein searching, however, all the information was quite old and mostly from synthesis chemistry papers. Usually, there was minimal information on methodology and quite fundamental information, e.g. the hydrolysis of methyl salicylate in aqueous solution, could not be located. Where possible, values can be calculated using available software. For the two parameters, photochemical oxidative degradation (Atkinson) and log Kow, calculated values were added to the summaries. In the case of hop -acids, however, the estimated value for log Kow was much higher than expected of the compound, as these cyclohexane triones are weak acids and mostly ionised at pH 7. In this project it was also necessary to refer to some secondary sources without any experimental details for some of the data. Furthermore, some contradictory data points were obtained for certain properties of cis-jasmone.

From the 91/414/EEC regulatory perspective, an active substance of known purity and specification is the entity to be listed on Annex 1 of the Directive and approved in plant protection products, and this particular material must be used in studies, or justifications made as to the acceptability of other test substances in supporting this commercial material. The registration process does require certain experimental data, and when conducted GLP is a stated requirement for all new safety studies under Directive 91/414/EEC.

It is clear from this project that some information relating to physical and chemical properties of the three candidate compounds are available from the public domain in a format that appears to satisfy today’s regulatory standards. The published data do provide a useful overview of the likely properties of each compound, but clarification may be requested on the exact nature and properties of the substances as well as the technical materials which will be commercialised (including whether there is more than one active component, as is the case for hop -acids). In particular it is possible that authorities would require information on impurities in the commercial material and test substances used in the dossier, especially where the data are key in supporting reasoned cases and risk assessments in other pivotal sections of the dossier.

Therefore, it may be appropriate in all three cases to conduct some up to date key studies on the active substances and commercial materials to current regulatory guidelines, thereby providing unequivocal physical/chemical properties data. Some data on the pure active substance may be accepted from alternative sources if sufficient details are provided and/or information comes from a reputable source e.g. a refereed journal.

It is difficult to justify commissioning new studies if published parameters are sufficient to allow a risk assessment to be conducted, and in particular if these are values which have been commonly accepted as genuine by scientists with long term experience in the particular area

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of research. The specific needs of the synthetic chemicals regulatory regime must be applied with pragmatism for these semiochemicals, allowing limited resources to be focused on the key issues.

This issue is considered to be an important point for further discussion with PSD, who are invited to consider the extent to which new data on the specific registerable material would be essentially required for these three compounds

Section 1 Further information

This section that refers to the field trial efficacy data and proposed use profile of the substance.

For none of these three materials are we in the position of demonstrating >90% control at a particular dose, but with all compounds there is a demonstrable effect on pest populations. For each material there were two reports ex. Rothamsted in the open literature that describe the effects in controlling pests (aphids or spider mites). In some cases it was difficult to abstract the actual numerical efficacy results from the published papers, as they were often presented in a graphical mode rather than as a table. PSD encourage applicants to submit raw data if using published papers in the dossier, but in these cases it would be rarely available. Applying current PSD regulatory requirements, these published reports alone would potentially be insufficient under current legislation and regulatory practice to support a product approval. Therefore further GEP efficacy trials plus the usual biological assessment overview dossier would be required as part of the Annex III of each submitted dossier. The need for trials conducted at ‘Officially Recognised’ testing sites would necessitate sub-contracting the work outside of Rothamsted Research, at additional cost. It is proposed that this is a key issue for discussion with PSD as to the extent to which the known and publicly documented efficacy of these candidates could be used to support a case for approval, potentially without an initial package of costly efficacy trials. Further comments on the issue of the relative costs of developing efficacy dossiers to progress these three compounds through to registration are provided in Appendix 2.

Commercial material safety data sheets were located for all three active substances.

The section also requires information on the proposed uses of active substances. As such a representative set of Good Agricultural Practice (‘GAP’) tables (dossier Document D-1) were compiled. Use scenarios are selected which have proven efficacy and known commercial interest, and they exemplify a range of approaches to address data requirements including justifications against data generation. Selection of these use scenarios was important to focus the data gap analysis and allow discussions to develop on the specific properties of the three compounds. If the compounds are proposed for use in scenarios other than these identified, it would be necessary to reassess these exposure scenarios and reconsider the data gaps accordingly.

For each compound, the scenarios were:

Methyl Salicylate: An unformulated volatile compound which occurs naturally and is released only into the air from impregnated plastic strips suspended around cereal crops

cis-Jasmone: A plant extract sprayed with a proprietary wetter using conventional hydraulic application methods to cereals

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Hop -acids: A plant extract in formulation which is applied to the crop from which it has been extracted (hops) using ultra low volume equipment

Section 2 Analytical methods

Analytical methods for the determination of the active substance in the technical material as manufactured were available for hop -acids, but, no supporting validation data were provided to confirm specificity, linearity or accuracy.

The main research effort at Rothamsted Research has been developing and using equipment and methods for identifying and analysing semiochemicals i.e. cis-jasmone and methyl salicylate using air-entrainment, gas chromatography (GC), GC-Mass spectrometry and Nuclear Magnetic Resonance (NMR). These methods will be used to validate and support the specification of the technical material at a time when a decision is taken to move to the commercial phase and to gain registration, i.e. when the specific details of the commercial materials are finalised.

No methods for the determination of residues in crops or the different environmental compartments (soil, water, air) are available for any of the three candidates. However, it is reasonable, and in line with standard regulatory practice, to assume that in the cases where scientific arguments can be provided to demonstrate no exposure, analytical methods would not be required.

Section 3 Toxicological and metabolism studies

More published data were found for methyl salicylate than for cis-jasmone and hop -acids. Only two reports (for methyl salicylate) were to GLP standard and the remainder varied much in quality. Again, in some instances it was considered necessary to include data from secondary sources with no methodology, as access could not be obtained to the original cited reports.

For most semiochemicals there is likely to be insufficient toxicology data of acceptable quality to allow Annex I inclusion if all the listed data requirements of 91/414/EEC are to be formally interpreted and satisfied. In this respect methyl salicylate, cis-jasmone and hop β-acids and are not exceptions.

The OECD Guidance for Registration Requirements for Pheromones and Other Semiochemicals4 has provided a useful rationale for considering reduced data requirements but some of the assumptions used to justify a reduction in data requirements do not necessarily apply to all semiochemicals. For example, application of cis-jasmone is by conventional spraying methods which is inconsistent with the application rate being ‘typically low and probably comparable to natural emissions’. As the commercial value of semiochemicals is unlikely to be enough to bear the cost of generating new data for endpoints such as carcinogenicity or reproductive toxicology, there is a need to consider whether alternative sources of information can be used in the approval process. For example, in aromatherapy treatment, jasmine oil is applied to the skin in a carrier oil.

4 OECD Environment, Health and Safety Publications. Series on Pesticides No. 12. Guidance for Registration Requirements for Pheromones and Other Semiochemicals Used for Arthropod Pest Control

16

Jasmine oil, purchased from a local chemist, was analysed using gas chromatography and was found to contain 3.5% cis-jasmone. It was calculated from aromatherapy instruction leaflets that the quantity of cis-jasmone applied to the skin was greater per square metre that the quantity of cis-jasmone applied in the field per square metre5.

Methyl salicylate has a variety of other approved uses and also occurs naturally, it was therefore selected for a more comprehensive evaluation than either cis-jasmone and hop β-acids. A combination of the available toxicology data, the opinion of expert groups who have approved methyl salicylate for other uses and information on current human exposure to methyl salicylate has been used to support values for the acceptable daily intake (ADI), acceptable operator exposure levels (AOEL) and the acute reference dose (ARfD).

Consideration was also given as to whether the low levels of exposure associated with the proposed use of methyl salicylate as a pesticide should be a factor that justified a reduced data set. This has not been taken into consideration in this instance because it was felt that Annex II, Section 3 should only be concerned with hazard and this should not be influenced by the likely exposure. It is recognised, however, that this is a debatable point for further discussion with PSD.

This point is noted in contrast to comments made in the approach for the environmental risk assessment below, which does allow the exposure profile to influence the range of non-target species tested because they occupy different environmental niches.

The use of the concept of a threshold of toxicological concern could also be considered in order to minimise generating new data. The threshold of toxicological concern (TTC) is a value below which there is a very low probability of appreciable risk to human health. The principle of the TTC is that a threshold can be applied to many substances in the absence of a full toxicological database. The concept of the TTC was developed by Munro (19906, 19967, 19998) as a result of a review of toxicity data of chemicals which were divided into three structural classes. This showed that a TTC could be assigned to each structural class; allocation of a chemical structure to a structural class therefore allows a TTC to be assigned. The TTC concept is used by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) to evaluate flavouring agents and also by the US Food and Drug Administration for consideration of indirect food additives. The TTC approach is also currently used in Europe in the evaluation of Food Additives, Processing Aids and Materials in Contact with Food, and a recent opinion on this topic has been published this year in the EFSA Journal9. Furthermore, in the context of European plant protection products’ regulation, the concept of a TTC is used in a late stage of the stepwise process for evaluation of significant metabolites in groundwater10.5 Rothamsted Research, in-house data, 20056 Munro, I.C. (1990) Safety assessment procedures for indirect food additives, an overview. Report of a workshop. Regulatory Toxicology and Pharmacology 12, 2-12.7 Munro, I.C., Ford, R.A., Kennepohl, E., Sprenger, J.G. (1996) Correlation of structural class with no-observed effect levels: a proposal for establishing a threshold of concern. Food and Chemical Toxicology 34, 829-867.8 Munro IC, Kennepohl E, Kroes R (1999). Application of a threshold of toxicological concern in the safety evaluation of certain flavouring substances. Food Chem. Toxicol. 37, 207-232.9 Opinion of the Scientific Panel on Food Additives, Flavourings, Processing Aids and Materials in contact with Food (AFC) on a request from the Commission related to Flavouring Group Evaluation 12: Primary saturated or unsaturated alicyclic alcohol, aldehyde, and esters from chemical group 7 (Commission Regulation (EC) No 1565/2000 of 18 July 2000) Adopted 23 February 2005. The EFSA Journal (2005) 208, 1-38.10 Guidance Document on the Assessment of the Relevance of Metabolites in Groundwater of Substances Regulated under Council Directive 91/414/EEC. SANCO/221/2000 –rev.10-final. 25 February 2003.

17

The European Chemical Substances Information System was checked for the three compounds11 and an IUCLID Dataset was found for methyl salicylate (CAS 119-36-8), thus indicating that in parallel, notifiers are defending the substance in the EU for a range of uses. The IUCLID Dataset will be compared against the current dossier documents.

The TTC is used by JECFA as part of decision tree and a decision tree approach has also been recommended recently by an expert group convened by the International Life Sciences Institute (ILSI). A simple decision tree approach that could be applied to semiochemicals is shown in Figure 1.

11 The European Chemical Substances Information System (ESIS) is found on the European Chemicals Bureau website http://ecb.jrc.it/

18

Figure 1

Decision tree approach using Threshold of Toxicological Concern (TTC)

No

Yes

Yes No

Yes

No

cis-Jasmone and hop β-acids and also have other approved uses and occur naturally. As was done for methyl-salicylate, and with the availability of further resource, the expert opinions

19

Estimate dietary and operator exposure

Estimate exposure to naturally occurring levels

Dietary/operator exposure > Natural exposure

No further work required on active substance

Identify structural class and assign TTC

Dietary/operator exposure > TTC

Consider what studies are needed

No further work required on active substance

Required data available from literature search

No further work required on active substance

Conduct studies

on which other uses of these compounds are based, along with the likely current human exposure to naturally occurring residues will be included to complete future regulatory submissions. It has been assumed at this stage that the information available will be sufficient to overcome the need for additional sub chronic, chronic and reproductive toxicity studies and this has been taken into account in the data gap analysis for these substances.

Section 4 Residues

From the literature search, we did not find long term residue studies following field application of the three materials. However, deposition studies conducted in-house at Rothamsted have assessed the amounts of material present on crops after application. It is expected that these data would be useful for inclusion in regulatory dossier(s).

The potential concern regarding residues is likely to mostly affect hop -acids and possibly cis-jasmone, as these two materials are sprayed. Assessment of levels on crops would be required and compared with natural emissions. It is possible that the high vapour pressure of cis-jasmone will reduce exposure in the ‘treated’ cereal crop thus allowing the presentation of scientific justifications for the absence of residues data.

For hop -acids, it is questionable whether it would be feasible to distinguish applied material from levels that would occur naturally in hops, thereby providing reasoned justification for not generating residues data. This would of course depend on timing, frequency and rates of application as well as estimating naturally occurring levels.

The requirement for a consumer risk assessment will not only depend on the potential exposure from residues on the consumable part of the crop but also on appropriate endpoints derived from the toxicological profile of the active substance and any relevant metabolites. Compounds such as hop -acids already have an FDA GRAS (Generally Regarded as Safe) notice status in the US. Overall, it is expected that reasoned arguments could be made to establish that consumers would not be at risk. Such arguments would include comparative calculations of the levels of hop -acids in the applied product compared with those in the hop crop at relevant growth stage(s).

Section 5 Fate and behaviour in the environment

Some soil degradation studies were found for methyl salicylate, albeit lacking in detail. No information was found on hop -acids or cis-jasmone. The route and significance of environmental exposure are considered key in determining the environmental fate data requirements for semiochemicals. In the case of methyl salicylate, which is intended to be suspended in the crop following impregnation onto materials such as plastic strips, the main route of environmental exposure is expected to be volatilisation into the atmosphere. Reasoned arguments can be made to demonstrate that exposure to soil and water will be negligible and data requirements on the fate and behaviour of this compound in soil and water may be waived. However, in the case of cis-jasmone and hop -acids, which are intended for outdoor spray application, potential routes of environmental exposure will include crop washoff / direct overspray to soil, spray drift / runoff to surface water bodies, possible leaching to groundwater and volatilisation into the atmosphere. The recommendations in this case would therefore be to conduct a series of key studies to provide information on the environmental fate and behaviour of these compounds in each environmental compartment. Data will also be required if there are any potentially relevant metabolites.

20

Bridging data from alternative sources may be acceptable if sufficient details are provided and the information is taken from a reputable source e.g. refereed publications. A good degree of similarity between the chemicals should also be demonstrated. In all cases, environmental fate and behaviour data requirements may also be waived if it can be demonstrated that the proposed application rates of the three candidate compounds are lower than or comparable to natural emissions. However, in reality it is questionable whether it would be feasible to make accurate measurements of naturally occurring background levels at given time points and for hop -acids in particular, it may not be technically possible to distinguish applied material from levels that would occur naturally in hops. Thus this would be a key consideration when deciding whether to use the ‘natural occurrence’ argument in justifications for the absence of studies in the package. Data may also be waived in circumstances where ecotoxicology data clearly indicate that there is no hazard to biota.

These compounds are not toxic at levels applied to the environment and the behavioural effects are known and documented. In behavioural studies, cis-jasmone was found to significantly repellent to a number of aphids while in contrast it was significantly attractive to aphid predators and parasitoids. cis-Jasmone dossier ref. IIA. 3.5.1/01. In field experiments, both in Sweden and UK, methyl salicylate reduced aphid colonisation significantly. Methyl salicylate dossier ref. IIA. 3.4.3/01. Again in bioassay, hop -acids was repellent to the two spotted spider mite and reduced feeding and oviposition, Hop -acids dossier ref. IIA. 3.5.1/01.

Section 6 Ecotoxicological studies

No information was found on hop -acids or cis-jasmone and very little on methyl salicylate (bees and earthworms only). There is currently, little specific regulatory guidance for the environmental risk assessment of semiochemicals, apart from the detailed guidelines for conventional pesticides provided under Council Directive 91/414/EEC (as detailed in Commission Directive 96/12/EC). The only relevant guidance is provided in OECD Guidance12. This is of particular relevance to pheromones13 and the requirements do not necessarily apply to all groups of semiochemicals (where more or less information may be required compared with pheromones).

The OECD guidance does however establish a number of important principals: the need to focus on the End Product and the possibility of reduced data requirements. In the case of semiochemicals, it is the circumstances in which they are used that are of particular importance to their potential risk to non-target organisms. The End Product is thus particularly relevant as it will determine the range and extent of non-target exposure, the expression of effects (e.g. in relation to the levels at which they occur, timing etc) and the context in relation to possible natural (background) exposure levels. The conventional assessment of toxicity i.e. a fixed property of a compound, the expression of which increases with exposure, is less appropriate.

Following on from this, by focussing on the actual risk from the use of a particular semiochemical, it may be possible to reduce the data requirements and this is demonstrated by

12 OECD Environment, Health and Safety Publications. Series on Pesticides No. 12. Guidance for Registration Requirements for Pheromones and Other Semiochemicals Used for Arthropod Pest Control. (ENV/JM/MONO(2001)12, 26-Feb-2002).13 Defined by OECD as: “Pheromones are semiochemicals produced by individuals of a species that modify the behaviour of other individuals of the same species (i.e. an intraspecific effect).”

21

the Straight-Chained Lepidopteran Pheromones (SCLPs)14. The OECD guidance points out that in such a case, the data requirements may be considerably reduced e.g. because of their known specificity and low toxicity to mammals. For this reason, the usual approach for conventional pesticides i.e. obtaining a standard package of toxicity data, which is then combined with an assessment of potential exposure in the overall risk assessment, needs to be applied in a flexible manner.

If the primary focus of any guidance for semiochemicals is on the actual environmental risk assessment, then the data requirements can be identified from this (rather than producing a definitive package of data for a standard risk assessment). This is particularly relevant given the potential diversity of semiochemicals (e.g. in terms of their mode of action, use patterns, natural occurrence etc). It is thus important first to consider all the available, relevant information for any specific semiochemical e.g. mode of action, target specificity, application technique, environmental fate profile, physical chemical properties etc. In this way, an initial assessment can be made of those non-target groups where there is a potentially significant risk e.g. avian/mammalian, aquatic, honey bees, non-target arthropods, soil-dwelling organisms etc. Particular attention can then be given to these groups and where necessary specific data generated to enable a more detailed assessment of the risk to be conducted. There must be reasonable confidence in the overall risk assessment and where key information is lacking e.g. in relation to specificity, mode of action, fate properties etc., additional data may be required. In contrast with the approach that must be followed for setting the acceptable daily intake (ADI) for use in human risk assessment, it is feasible in the environmental risk assessment to allow the exposure profile of the proposed product to influence the range of non-target species which are tested.

As part of this process, it may be possible to identify a sequential approach to the risk assessment process, using a formalised decision-tree (Figure 2) to enable a decision to be made on what level of input is required for a particular semiochemical. An example of this is provided by the three candidate semiochemicals identified in this project: cis-jasmone, methyl salicylate and hop -acids:

Methyl salicylate

This is a naturally occurring compound that is involved in host plant location by Rhopalosiphum padi. Application to the summer host results in repellency and this activity is also expressed with other aphid species. It is intended for use in a very specific manner i.e. by suspension in the crop following impregnation onto materials such as plastic strips. However, exposure will be more widespread but transient as the main route will be by volatilisation into the atmosphere. Exposure to soil and water will thus be negligible (provided there is no wash-off from the strips). A number of factors would thus suggest that the risk will be low e.g. localised distribution with only low, transient exposure on a more widespread basis; natural occurrence (consideration should be given to natural levels of emission); mode of action (linked to specificity). Particular attention would be given to honey bees and non-target arthropods (taking into account any available information e.g. from published sources). On this basis, data requirements could be very limited.

14 Defined by OECD as: “Straight-chained lepidopteran pheromones (SCLPs) are a group of pheromones consisting of unbranched aliphatics having a chain of nine to eighteen carbons, containing up to three double bonds, ending in an alcohol, acetate or aldehyde functional group. This structural definition encompasses the majority of known pheromones produced by insects in the order Lepidoptera, which includes butterflies and moths.”

22

cis-Jasmone

cis-Jasmone is a natural elicitor of plant defence against insects. However, the proposed application involves ground-based spraying with hydraulic nozzles, so that non-target organism exposure will be widespread e.g. soil-dwelling organisms, off-field (aquatic, non-target plants, non-target arthropods), honey bees, birds/mammals. This exposure may be significant in relation to natural (background) levels in the relevant environmental compartments and so would need to be considered further. Additional information on the mode of action could be considered but it is likely that the data requirements in such a case would be more extensive. However, given the likely transient, volatile, nature of this compound (to be confirmed by the Fate and Behaviour assessment) it is likely that the focus of the risk assessment would be mainly short-term (acute).

Hop β-acids

A natural hop product, which would be applied on to hops using a rotary atomiser (ULV). Consideration would need to be given to the levels of exposure in relation to the natural levels within the target crop. Also, consideration needs to be given to off-crop exposure, although it might be possible to address this by the application technique (ULV will limit drift but use of spray tunnels would greatly reduce this aspect). This example would definitely need further detailed consideration before characterising the potential extent and level of risk and could perhaps result in an intermediate level of data requirements.

23

Figure 2Decision tree approach for environmental risk assessment

Yes No

No

Yes

Yes

No

Classification and labelling

24

Identify relevant available information e.g. mode of action, application technique etc

Consider if exposure and effect likely

Environmental exposure > Natural exposure

No further work required on active substanceEstimate

environmental exposure

Estimate exposure to naturally occurring levels

Consider what additional information is needed

No further work required on active substance

Required information available from literature search

No further work required on active substance

Conduct studies

The current project was conducted with reference to the risk assessment of active substances and plant protection products containing semiochemicals. It is acknowledged that other EU legislation also requires active substances and formulations to be classified with regard to their hazard profile and labelled accordingly. Studies may also be required to allow classification, and in some cases classification of formulations can be calculated based on properties of the co-formulants.

4. Assessment of costs required to complete the packages to a standard suitable for regulatory purposes

The available published studies were summarised in each M-II ‘Tier 2’ dossier document to first draft stage and reference lists were compiled. Then a ‘data gap analysis’ was conducted for each compound by experts in each of the following areas: Physical Chemical Properties, Analytical Chemistry, Mammalian Toxicology, Residues, Environmental fate and Behaviour and Ecotoxicology. Experts referred to relevant guidance, the summaries of the published papers obtained and the associated use scenarios. A spreadsheet of the key outstanding studies required to complete each dossier to registration standard was then prepared. The data gaps were identified by relevant EU Annex point numbers and cost estimates to conduct each study listed were obtained. The cost estimates for these studies were an average price based on JSC’s knowledge of reputable GLP-accredited contract research organisations at the time of writing (2004-2005).

This costings exercise was valuable in an essential part of the process where a biopesticide company must develop robust business cases to justify product development and regulatory costs, by comparing such costs with the potential market for the plant protection product.

Recent estimates by Agrow15 are that biopesticides hold a relatively small proportion (1.15% or $330 million) of the global pesticides market. The total EU market for biopesticides is estimated at $97 million16. It has been assumed to date that the costs to develop a registration package for a new biopesticide would be in the region of $1-1.5 million.

This project considered the degree to which the registration costs would be offset by using data and information gained through literature searching. Importantly, it is stressed that cost comparisons should be interpreted with caution and the resultant figures be taken in context. The figures provided can give an idea of regulatory costs outstanding for the three compounds as a comparative exercise.

The types of costs required to complete the dossiers ready for submission fall into in the following categories:

1. Key data gaps where there was a requirement perceived for new studies to complete the Annex II (active substance) data package;

2. Studies that are required to complete the Annex III (formulated product) data package. As there is not yet a formulation specification associated with the three actives, a nominal cost based on a ‘standard’ Annex III package has been assumed;

3. A cost for study monitoring which is usually estimated at 15-20% of the study costs, and would ensure studies are conducted correctly and in a timely fashion;

4. Efficacy trials program and the overall Biological Assessment Dossier preparation;

15 IBC AgChem Forum, September , 2004 16 Frost and Sullivan (2001)

25

5. Cost estimate to include the new studies in the dossier documents and complete them to comply with the necessary OECD dossier format guidance;

6. Costs to translate published papers into English;7. Regulatory application fees to UK PSD, as applicable under the current pilot scheme for

alternative control measures (all three actives defined in the pheromones category).

We have not included the costs of ongoing regulatory support after dossier submission but this must not be overlooked, as the lack of dedicated resource in this area is one suggested reason for slow progress when applicants are asked to deal with PSD requests for further information during evaluation.

The resultant costings information obtained from this part of the exercise are presented in Appendix 2. There are some important points to be considered during the interpretation of the figures in these tables:

Scientific dossier studies are always generated using a stepwise approach. Therefore it is not possible to predict with full certainty whether any further studies would be triggered after conducting those identified in this initial list. A possible example would be if significant metabolites are elucidated following degradation of the active substances in environmental compartments.

In view of the potential natural source and occurrence of these semiochemicals, there is a case for conducting studies in sequence as some will may depend on others. An example would be to first conduct basic studies on the environmental effects of the products i.e. ecotoxicological studies, in order to determine whether there is a hazard to biota. If not, then PSD may consider that it would be possible that the requirement for extensive environmental fate and behaviour studies could be waived.

The cost estimates for the efficacy package are based on a simple commercial product label with one main control claim and one crop17. Efficacy trials for biopesticides often have more complex design than those for chemical products which also has a cost implication.

Conservative estimates of individual study costs were made which could be refined with further information on the commercial technical material and the co-formulants to be used in plant protection products. Also, contract labs could be selected based on price reductions for batches of studies. Therefore it is noted that the overall cost figure may have compounded the conservatism in this estimation. In some cases, studies may be conducted in-house, but GLP is required for all safety studies under Directive 91/414/EEC.

Knowledge of the nature of the formulation components, when established, may allow further waivers or introduce further studies into the list of requirements.

Prices estimated are exclusive of VAT.

Specific observations on the costings for each substance (Appendix 2) are as follows.

Methyl salicylate

It is shown that this dossier of the three would be least costly to complete to a standard suitable for regulatory submission to UK PSD. The two main reasons why this is the case for 17 We are grateful to Agrisearch UK Ltd. for assisting with these cost estimates

26

methyl salicylate are; Firstly, the availability of existing toxicological reviews and regulatory documents on the methyl salicylate and related compounds which support acceptable use. Secondly, the compound is not formulated so the Annex III package would be limited to studying the release of the compound from its plastic strip applicator system. The costs to generate the efficacy package would constitute approximately 38% of the cost of the outstanding regulatory package.

cis-Jasmone

cis-Jasmone gives a higher cost estimate for dossier completion that methyl salicylate which is partly due to the sparse information found in the literature. This is also due to the proposed method of application, conventional hydraulic spraying, which would require detailed regulatory evaluation. This is coupled with the efficacious dose being above naturally occurring or natural emission levels. In particular a large package of studies in the Environmental Fate and Behaviour and Ecotoxicology areas emerges as an outstanding requirement for cis-jasmone. It is hoped that further discussions with PSD and application of the proposed approaches discussed above at Section 3.2 could potentially lead to a less extensive list of key data gaps. The costs to generate the efficacy package would constitute approximately 14% of the cost of the outstanding regulatory package.

Hop -acids

The estimated costs for completing the hop -acids dossier were comparable to that for cis-jasmone. Despite hops acids being applied to hops which would probably require a simple human dietary assessment; the costs were partly due to the complex nature of the active substance. There are a number of active components in the technical material, on which some degree of independent analysis and quality assurance would be required. Again in this case, a large number of Environmental Fate and Behaviour and Ecotoxicology data requirements are triggered, but these may be refined by considering whether modifications in the proposed use profile would reduce the off-crop exposure e.g. use in spray tunnels. The costs to generate the efficacy package would constitute approximately 13% of the cost of the outstanding regulatory package.

27

Appendix 1 - Use scenarios used as the basis of the data gap analysis

28

Methyl salicylateDetails of Intended Uses (GAP information)

Crop Member Product F Pests or Formulation Application Application rate per treatment PHI Remarks and/ or situation (a)

State or Country

name G or I

(b)

Group of pests controlled (c)

Type

(d-f)

Conc. of as (i)

method kind (f-h)

growth stage &

season (j)

number

min max(k)

interval between

applications (min)

kg as/hl

min max

water l/ha

min max

kg as/ha

min max

(days)

(l) (m)

WheatOat

Barley

UK N/A F Sitobion avenae, Metapolophium dirhodiumRhopalosiphum padi

(1)(2)(3)

(4) Volatilisation from(1)(2)(3)

mid seasongrowth stage 35

1 none N/A N/A 20mgperdayper

10

(1) Impregnated plastic strips suspended in the crop.(2) Impregnated on to sponge inside a sealed polythene bag.(3) Impregnated in to wax pellets in mesh string bags and used in glasshouses.(4) Impregnation can range between 10µl to 1.5mls to provide a release rate of 0.5mg/day to 150mg/day. Typical is 20mg/day.

29

cis-JasmoneDetails of Intended Uses (GAP information)

Crop Member Product F Pests or Formulation Application Application rate per treatment PHI Remarks and/ or situation (a)

State or Country

name G or I

(b)

Group of pests controlled (c)

Type

(d-f)

Conc. of as (i)

method kind (f-h)

growth stage &

season (j)

number

min max(k)

interval between

applications (min)

kg as/hl

min max

water l/ha

min max

kg as/ha

min max

(days)

(l) (m)

Wheat UK N/A F Metopolophium

dirhodum,Rhopalosi

phum padi,

Sitobion avenae

(3) (1)(2)

mid seasongrowth stage 35

1 min2 max

2 to 3 weeks 200 50g 2 weeks

(1) Conventional hydraulic sprayer(2) Impregnated in to wax pellets and used as a point source(3) Applied in 200l of water with 0.1% Agral wetting agent.

30

Hop -acidsDetails of Intended Uses (GAP information)

Crop Member Product F Pests or Formulation Application Application rate per treatment PHI Remarks and/ or situation (a)

State or Country

name G or I

(b)

Group of pests controlled (c)

Type

(d-f)

Conc. of as (i)

method kind (f-h)

growth stage &

season (j)

number

min max(k)

interval between

applications (min)

kg as/hl

min max

water l/ha

min max

kg as/ha

min max

(days)

(l) (m)

Hops UK N/A F Two-spotted spider mite (Tetranychus urticae Koch)

(1) Rotary atom-iser

(ULV)

1.0m high to 4.0m high

2-3 2 - 3 weeks 26water

ethanolbeta acidsmix

2.6 2weeks

(1) The formulation concentration is 100g/l beta acids, 800g/l ethanol and 100g/l water. This is applied as an undiluted formulation.

31

Appendix 2 Summary of costs outstanding to complete the packages for regulatory submission.

32

Methyl salicylate

Section Point Study Estimated costIIA, Section 1 PhysChem Active substance*data available II, 2 Melting, boiling point £450

II, 2 Vapour pressure and volatility £2,000II, 2 Physical state, colour, odour £100II, 2 UV/Vis, IR, NMR, MS spectra £3,500II, 2 Solubility (water* and solvents) £2,500II, 2 Dissociation constant £500II, 2 Partition coefficient n-octanol/water £2,275

Technical materialII, 2 Relative density £450II, 2 Physical state, colour, odour £100II, 2 Flammability, auto flammability £550II, 2 Flash point £550II, 2 Explosive properties £1,700II, 2 Surface tension £470II, 2 Oxidising properties £1,500

IIA, Section 2 Analytical Methods II, 4 For a.s. and impurities in technical £15,000

IIA, Section 3 Toxicology II, 5 Not considered necessary at this stage £0

IIA, Section 4 Residues II, 6 Not considered necessary at this stage £0

IIA, Section 5 Environmental Fate II, 7 Atkinson calculation (est. photochemical degradation) £1,000

IIA, Section 6 Ecotoxicology II, 8 Honey bee £3,300II, 8 Fish £4,200II, 8 Daphnia £4,060II, 8 Algae £4,200

IIIA, Section 1 PhysChem III, 2 Physical-chemical properties - final 'VP' product on strips £17,400III, 2 Two year storage stability study - final 'VP' product on strips £8,600

IIIA, Section 2 Analytical Methods III, 5 Analytical method - final 'VP' product on strips £5,000

IIIA, Section 3 Toxicology III, 7 Probably not required - same as active substance £0

IIIA, Section 4 Residues IIIA, 8 Probably not required - same as active substance £0

IIIA, Section 5 Environmental Fate III, 9.1-9.3 PECs in soil, groundwater & surface water are probably not required as it can be argued that the main route of environmental exposure is volatilisation into the atmosphere

£0

IIIA, Section 6 Ecotoxicology IIIA, 10 Probably not required - same as active substance £0

Total Annex II plus Annex III studies £68,080Study monitoring at 15% £10,212

IIIA, Efficacy Efficacy trials £55,000Crop destruction and assessment of yield/grain quality £10,000Simple biological dossier £6,000

OECD dossier A to O Completion of dossier preparation work £25,000

Translation of published papers £2,000

PSD application fee Pilot project exercise (fee 2004-2005) £13,000

TOTAL REMAINING COST £189,292

33

cis-Jasmone

Section Point Study Estimated costIIA, Section 1 PhysChem Active substance*data available II, 2 Melting, boiling point £450

II, 2 Vapour pressure and volatility £2,000II, 2 Physical state, colour, odour £100II, 2 UV/Vis, IR, NMR, MS spectra £3,500II, 2 Solubility (water and solvents) £2,500II, 2 Partition coefficient n-octanol/water (flask shake) £2,275

Technical materialII, 2 Relative density £450II, 2 Physical state, colour, odour £100II, 2 Flammability, auto flammability £550II, 2 Flash point* £550II, 2 Explosive properties £1,700II, 2 Surface tension £470II, 2 Oxidising properties £1,500

IIA, Section 2 Analytical Methods IIA, 4 For a.s. and impurities in technical £15,000IIA, Section 3 Toxicology IIA, 5 Acute oral £1,125

IIA, 5 Acute dermal £1,125IIA, 5 Inhalation £10,000IIA, 5 Skin irritation £1,475IIA, 5 Eye irritation £1,375IIA, 5 Skin sensitisation (LLNA) £1,975IIA, 5 Ames test £2,650IIA, 5 Mouse lymphoma assay £11,000IIA, 5 Chromosome aberration £14,250

IIA, Section 4 Residues IIA, 6 Dependent on toxicological profile £0IIA, Section 5 Environmental Fate IIA, 7 Aerobic soil metabolism £60,000

IIA, 7 Adsorption/desorption £30,000IIA, 7 Aerobic aquatic metabolism £50,000IIA, 7 Soil photolysis £36,000IIA, 7 Aqueous photolysis £35,000

IIA, Section 6 Ecotoxicology IIA, 8 Avian dietary studies - 2 species £12,600IIA, 8 Honey Bee £3,300IIA, 8 Soil Micro-organisms £4,500IIA, 8 Fish £4,200IIA, 8 Daphnia £4,060IIA, 8 Algae £4,200IIA, 8 Earthworms £2,400

IIIA, Section 1 PhysChem III, 2 Physical-chemical properties - formulation £17,400III, 2 Two year storage stability study - formulation £8,600

IIIA, Section 2 Analytical Methods III, 5 Analytical method - formulation £5,000IIIA, Section 3 Toxicology III, 7.1.1 Acute oral toxicity £1,125

III, 7.1.2 Acute dermal toxicity £1,125III, 7.1.4 Skin irritation £1,375III, 7.1.5 Eye irritation £1,475III, 7.1.6 Skin sensitisation (LLNA) £1,975

IIIA, Section 4 Residues IIIA, 8 Not required £0IIIA, Section 5 Environmental Fate III, 9.1-9.3 PECs in soil, groundwater & surface water £2,000IIIA, Section 6 Ecotoxicology III, 10.2.1 Fish acute – 2 species £13,000

III, 10.2.1 Daphnia – 48-hr EC50 £4,000III, 10.2.1 Algal inhibition – 72-hr EC50 £4,200III, 10.2.1 Lemna – 7 day EC50 £10,500III, 10.4.1 Honey bees – acute oral and contact LD50 £3,300III, 10.5.1 Non-target arthropods - 2 species £14,000III, 10.6.1 Earthworm - 14-day LC50 £2,400III, 10.8 Seedling emergence £15,000III, 10.8 Vegetative vigour £16,500

Total Annex II plus Annex III studies £445,355Study monitoring at 15% £66,803

IIIA, Efficacy Efficacy trials £70,000

34

Crop destruction and assessment of yield/grain quality

£10,000

Simple biological dossier £6,000OECD dossier A to O Completion of dossier preparation work £30,000Translation of published papers £2,000PSD application fee Pilot project exercise (fee 2004-2005) £13,000

TOTAL REMAINING COST £628,158

35

Hop -acids

Section Point Study Estimated costIIA, Section 1 PhysChem Active substance*data available II, 2 Melting, boiling point £450

II, 2 Vapour pressure and volatility £2,000II, 2 Physical state, colour, odour £100II, 2 UV/Vis, IR, NMR, MS spectra £3,500II, 2 Solubility (water and solvents) £2,500II, 2 Dissociation constant £500II, 2 Partition coefficient n-octanol/water (flask shake) £2,275

Technical materialII, 2 Relative density £450II, 2 Physical state, colour, odour £100II, 2 Flammability, auto flammability £550II, 2 Flash point £550II, 2 Explosive properties £1,700II, 2 Surface tension £470II, 2 Oxidising properties £1,500

IIA, Section 2 Analytical Methods IIA, 4 For a.s. and impurities in technical £15,000IIA, 4 Air and water methods (spray application) £10,000

IIA, Section 3 Toxicology IIA, 5 Acute oral £1,125IIA, 5 Acute dermal £1,125IIA, 5 Inhalation £10,000IIA, 5 Skin irritation £1,475IIA, 5 Eye irritation £1,375IIA, 5 Skin sensitisation (LLNA) £1,975IIA, 5 Ames test £2,650IIA, 5 Mouse lymphoma assay £11,000IIA, 5 Chromosome aberration £14,250

IIA, Section 4 Residues IIA, 6 Dependent on toxicological profile £0IIA, Section 5 Environmental Fate IIA, 7 Aerobic soil metabolism £60,000

IIA, 7 Adsorption/desorption £30,000IIA, 7 Aerobic aquatic metabolism £50,000IIA, 7 Soil photolysis £36,000IIA, 7 Aqueous photolysis £35,000IIA, 7 Atkinson calculation (est. photochemical

degradation) for 4 active components£4,000

IIA, Section 6 Ecotoxicology IIA, 8 Avian dietary studies - 2 species £12,600IIA, 8 Honey Bee £3,300IIA, 8 Soil Micro-organisms £4,500IIA, 8 Fish £4,200IIA, 8 Daphnia £4,060IIA, 8 Algae £4,200IIA, 8 Earthworms £2,400

IIIA, Section 1 PhysChem III, 2 Physical-chemical properties - formulation £17,400III, 2 Two year storage stability study - formulation £8,600

IIIA, Section 2 Analytical Methods III, 5 Analytical method - formulation £5,000IIIA, Section 3 Toxicology III, 7.1.1 Acute oral toxicity £1,125

III, 7.1.2 Acute dermal toxicity £1,125III, 7.1.3 Acute inhalation (waiver possible) £10,000III, 7.1.4 Skin irritation £1,375III, 7.1.5 Eye irritation £1,475III, 7.1.6 Skin sensitisation (LLNA) £1,975

IIIA, Section 4 Residues IIIA, 8 Not required £0IIIA, Section 5 Environmental Fate III, 9.1-9.3 PECs in soil, groundwater & surface water £4,000IIIA, Section 6 Ecotoxicology III, 10.2.1 Fish acute – 2 species £13,000

III, 10.2.1 Daphnia – 48-hr EC50 £4,000III, 10.2.1 Algal inhibition – 72-hr EC50 £4,200III, 10.2.1 Lemna – 7 day EC50 £10,500III, 10.4.1 Honey bees – acute oral and contact LD50 £3,300III, 10.5.1 Non-target arthropods - 2 species £14,000III, 10.6.1 Earthworm - 14-day LC50 £2,400III, 10.8 Seedling emergence £15,000III, 10.8 Vegetative vigour £16,500

Total Annex II plus Annex III studies £471,855

36

Study monitoring at 15% £70,778IIIA, Efficacy Efficacy trials £72,000

Crop destruction £10,000Simple biological dossier £6,000

OECD dossier A to O Completion of dossier preparation work £30,000Translation of published papers £2,000PSD application fee Pilot project exercise (fee 2004-2005) £13,000

TOTAL REMAINING COST £660,633

37